Photoactivation-based microscopy provides nanometer resolution

Putting into practice a microscopy scheme proposed a decade ago, researchers have imaged
fluorescent proteins within cells with 2- to 25-nm resolution. That is far below
the classical resolution limit of half a wavelength, which translates, for example,
to a few hundred nanometers in the visible.

The investigators, led by Eric Betzig and Harald
F. Hess of Howard Hughes Medical Institute’s Janelia Farm Research
Campus in Ashburn, Va., developed the technique, dubbed photoactivated localization
microscopy.

The key to achieving the resolution is that the
imaged fluorophores, which are a small subset of the total fluorescent molecules
present, are scattered across the sample, and the molecular signal is much larger
than any background noise. Thus, in every frame, the
fluorescent molecules that are visible are far enough from each other that their
diffraction-limited images, which are about 200 nm wide, do not overlap.

These isolated approximations of the ideal point
spread function of the fluorescence originating from a particular molecule allow
the center of each spot to be determined more precisely, with the error decreasing
by the square root of the number of photons captured. Because well more than 10,000 photons can
be detected from a single fluorophore before it photobleaches and disappears from
view, the location uncertainty for a molecule can be cut a hundredfold with this
technique.

Besides offering higher resolution than that possible
with other optical methods, the approach offers contrast-related benefits when compared
with electron microscopy.

To perform the technique, the researchers
used photoactivated fluorescent proteins that were expressed by the cell that they
studied. Thus, there was no need for the preparation
required for electron microscopy, which can sometimes disturb the sample.

In addition, Betzig noted, the method is more
discriminating in its labeling than that used in electron microscopy. “The
[photoactivated fluorescent proteins] are produced within the cell bound to only
a single target protein of interest and, therefore, give a very detailed map of
the spatial distribution of only that protein,” he said.

As described in the Aug. 10 edition
of Sciencexpress, the researchers serially activated
the fluorescent proteins using a brief pulse from a Coherent Inc. diode laser at
405 nm. They chose the intensity and duration of the pulse so that only sparse fields
of individually resolvable single molecules were activated. They then used a 561-nm
laser from Lasos Lasertechnik GmbH to image each fluorescent protein in turn until
the proteins photobleached. They captured the images using a cooled electron-multiplying
CCD camera from Andor Scientific, fitting the observed profile at each location
to the ideal 200-nm-wide point spread function.

In building up a single image of a thin section
of a cell or of the interface between a whole cell and the substrate on which it
rested, they repeated the process of activation and photobleaching as many as 100,000
times. Capturing a frame typically took between a half second and a second, which
meant that acquiring a complete image stack took from two to 12 hours.

Mitochondria (red) as revealed by photoactivated localization
microscopy are overlaid on a transmission electron microscopy image of
the same region.
Worried about nanometer-scale shifts
during these long data runs, the scientists built a custom microscope head designed
to reduce thermal and mechanical drift by minimizing the optical path length between
the sample and the objective lens. They added 50-nm gold beads to the sample and
localized their position in every frame to compensate for any drift, an approach
that Betzig said worked well. “These fiducial markers were so effective that
we can now contemplate applying photoactivated localization microscopy to commercial
microscopes of lower mechanical and thermal stability,” he said.

In demonstrations, the researchers
imaged specific target proteins in lysosomes and mitochondria. They visualized the
former via expression of the lysosomal transmembrane CD63, while they imaged the
latter using a fluorophore-tagged oxidase import sequence. They compared both with
transmission electron microscopy images, which showed a high degree of correlation
to those acquired with photoactivated localization microscopy. They also imaged
various targets in fixed whole cells, which again showed that the technique compared well with electron microscopy.
Resolution limitations of conventional optical microscopes lead to
blurry images of small cellular components such as mitochondria (top level). However,
if specific proteins are tagged with fluorescent molecules that can be activated
one at a time (between top and second level), the positions of the molecules be
can determined with much greater precision. Their positions then can be used to
assemble a photoactivated localization microscopy image (second level) at a resolution
comparable to that of an electron microscope (bottom level), with the added benefit
of being specific to the target protein.
Downsides of the approach are that
it suffers from long data collection times and that the photoactivated proteins
do not all produce the same number of photons. As a result, the localization precision
varies and, therefore, the highest-resolution image is the one with the fewest number
of labeling fluorophores.

Betzig noted that data collection might
be sped up by boosting the laser excitation power, lessening the time spent per
single molecule, or perhaps by increasing the number of activated molecules per
frame. It is unclear how much they can reduce the total time, but they hope to get
as low as five minutes for a single image.

As for the localization problem, oxygen-scavenging
techniques might increase the number of photons per molecule. There also is the
possibility of using more photostable activatable labels or other approaches that
could amplify the photon flow.

Betzig said that he saw photoactivated
localization microscopy as a research tool in cell biology, in particular, nanoscale
protein interactions that drive cellular functions. However, he added that more
work is required before it can fulfill that role.

That is one reason why the investigators
are planning to develop a multilabel version of their technique as well as to find
ways to increase the data acquisition speed. Another goal is to improve the ease
of use, turning the system into one that biologists could use routinely. “If
we get to that point, commercialization is a definite possibility,” Betzig
said.